The present invention relates to a method for producing a cholesterol-reduced substance, a cholesterol-reducing composition and a novel cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostane-3-one dehydrogenase for using the above-mentioned purpose.
It is widely known that excess intake of food having high cholesterol content increases the amount of cholesterol in serum and that high cholesterol content in serum is a significant factor in heart diseases. Therefore, processing techniques are required for selectively reducing the amount of cholesterol in food without deteriorating the quality of the food.
Among techniques for reducing the amount of cholesterol in food, a method is known that decomposes cholesterol with microorganisms (Japanese Laid-Open Patent Publication No. 267231/88) as a biochemical technique; however, this method produces by-products, therefore, it is not a safe method. Further, a method in which cholesterol is converted to epicholesterol by using an enzyme is known (WO93/25702).
Beitz, et al., U.S. Pat. No. 4,921,710 describes a method for converting cholesterol to coprostanol by using a cholesterol reductase derived from plants, and suggests a method for converting cholesterol to coprostanol by using a cholesterol reductase derived from bacteria such as Eubacterium species ATCC 21408. Also, Beitz, et al., U.S. Pat. No. 5,436,004 describes that the conversion ratio from cholesterol to coprostanol as 0.01% when a cream is treated using the above-mentioned enzyme derived from plants (see, column 5, table 1). However, such a low conversion ratio to coprostanol can not be admitted as practical level.
EYSSEN, British patent 1237483 describes that Eubacterium species bacterium separated from feces of rats reduces cholesterol to coprostanol, and also, in EYSSEN, Biochemica et Biophysica Acta, 348,279-284 (1974), it is estimated that the bacterium reduces cholesterol to coprostanol via 4-cholesten-3-one.
Beitz, et al., Applied Microbiology and Biotechnology 43, 887 (1995) describes that Eubacterium species bacterium (ATCC 51222) converts cholesterol in micelle to coprostanol, that 4-cholesten-3-one and trace amounts of coprostane have been detected in the conversion process, and that the reduction mechanism of cholesterol using the above-mentioned bacterium might be studied after pure preparations of the cholesterol reductase are obtained.
However, up to now, it has not been confirmed that cholesterol in a substance containing cholesterol is converted via 4-cholesten-3-one and coprostane-3-one to coprostanol by utilizing enzymatic action of cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase, coprostane-3-on dehydrogenase with using a coenzyme AND (P) and AND (P) H, and enzymes which convert cholesterol in a substance containing cholesterol to coprostanol via 4-cholesten-3-one and coprostane-3-one, respectively, have not been isolated from bacterium which reduces cholesterol, and in addition, no report have been proposed in which food is treated with these converting enzymes and microorganism containing the same.
It is known that cholesterol dehydrogenase derived from Nocardia, Alcaligenes, Proteus, which has optimum pH of around 9.0, requires AND (P) as a coenzyme, and is used for a quantitative determination of cholesterol (Japanese Post-Examined Patent Publication No.18064/90), however, this enzyme exhibits low activity at neutral pH, so that it can not be admitted as practical for food treatment.
Testing has been tried to obtain 4-cholesten-3-one dehydrogenase from the feces of a rat, however, the dehydrogenase is rapidly deactivated and can not be purified in this method, therefore, the method can not be admitted as practical (European J. of Biochemistry 37, 143 (1973)).
It is known that treatment of food with a phospholipase, protease and lipase accelerates conversion by a cholesterol oxidase (Japanese Laid-Open Patent publication No.76311/93), however, effect in enzymatic conversion of cholesterol to coprostanol is not known yet.
It is known that meat contains AND (H) (Journal of Food Science 37, 612 (1972)), and that nicotinamide inhibits decomposition of AND (Archives of Biochemistry and Biophisics 156, 143 (1973)). However, addition of nicotinamide in enzymatically converting cholesterol to coprostanol is not known.
It is known that an introduction of the cholesterol oxidase gene into lactic acid bacteria for decomposing food cholesterol (Applied Microbiology and Biotechnology 37, 330 (1992)).
As an applied example of these enzymes, the above-mentioned Beitz, et al., U.S. Pat. No. 5,436,004 suggests a treating method for reducing the amount of cholesterol in serum in which a cholesterol reductase derived from plants is orally administered. Further, it is reported that if a bacterium (ATCC 51222) which reduces cholesterol is orally administered to a rabbit suffering from hypercholesterolemia, the cholesterol level in serum decreases (Letters in Applied Microbiology 20, 137 (1995)).
Since absorbability of coprostanol through the intestinal tract is very low (American J. of Physiology 251, G495 (1986)), conversion of cholesterol to coprostanol is effective as a cholesterol reducing method. A practical method is not known for producing a cholesterol-reduced substance which reduces cholesterol by enzymatically converting cholesterol in food via 4-cholesten-3-one and coprostane-3-one to coprostanol. Also, a method for producing a practical enzyme which can be used in the above-mentioned production method is not known.
The object of the present invention is to provide a novel method for producing a practical cholesterol-reduced substance in food and feed. Another object of the present invention is to provide a novel cholesterol dehydrogenase having optimum pH in neutral pH range which is suitable for the above-mentioned practical method for producing a cholesterol-reduced substance, a novel 4-cholesten-3-one dehydrogenase and coprostane-3-one dehydrogenase having an optimum pH in a weak acidic pH range possessed by meat, and microbial cells and a treated material thereof containing these enzymes. A further object of the present invention is to provide a cholesterol-reducing composition which reduces cholesterol level in serum, comprising the above-mentioned three kinds of novel enzymes or comprising microbial cells containing these novel enzymes.
The present inventors have screened various microorganisms such as stock microorganisms mainly including 300 kinds (species categories) of actinomycetes, fungi and bacteria, aerobic bacteria separated from 100 kinds of various soils, and anaerobic bacteria from 7 feces samples of humans, 8 feces samples of mammals other than human and 9 feces samples of birds, to find a cholesterol reduction converting enzyme group having optimum pH in a neutral pH range or weak acidic range for solving the above-mentioned problems, and have intensively examined a culture medium for enzymatic activity detection and a method for recovering microbial cells, as a result, have accomplished the present invention.
Namely, the present invention relates to a process for producing a cholesterol-reduced substance and a method for reducing the amount of cholesterol, wherein cholesterol in a substance containing cholesterol such as meat, egg, milk, seafood and cooked processed food containing the same, or feed for animals, poultry and pisciculture, and the like, is treated with three kinds of enzymes consisting of cholesterol dehydrogenase having optimum pH in neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase having optimum pH in a weak acidic range or microbial cells containing these enzymes, to convert cholesterol to coprostanol for reducing the amount of cholesterol.
Further, the present invention relates to the above-mentioned three kinds of enzymes consisting of cholesterol dehydrogenase having optimum pH in a neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase having optimum pH in weak acidic range, produced by a strain belonging to Eubacterium, a cholesterol-reducing composition comprising these three kinds of enzymes, and a cholesterol-reducing composition comprising a strain belonging to Eubacterium which produce this enzyme group.
Further, the present invention relates to a process for producing a cholesterol-reduced substance and a method for reducing the amount of cholesterol wherein nicotinamide, phospholipase and nicotinamide, or a phosphate ion are added when cholesterol in a cholesterol-containing substance is converted to coprostanol by utilizing the above-mentioned enzyme group.
The process for producing a cholesterol-reduced substance of the present invention will be described in detail below.
In the present invention, examples of the cholesterol-containing substance include meat, egg, milk, seafood and cooked processed foods containing the same, or feeds for animals, poultry and pisciculture, and the like, but are not limited to them providing the substance containing cholesterol.
In the present invention, the cholesterol dehydrogenase is an enzyme conducting the following reaction.
Cholesterol+AND(P) xe2x86x924-cholesten-3-one+AND(P)H
Further, in the present invention, the 4-cholesten-3-one dehydrogenase is an enzyme conducting the following reaction.
4-Cholesten-3-one+AND(P)Hxe2x86x92coprostan-3-one+AND(P)
Similarly, in the present invention, the coprostan-3-one dehydrogenase is an enzyme conducting the following reaction.
Coprostan-3-one+AND(P)Hxe2x86x92coprostanol+AND(P)
In the present invention, the term xe2x80x9coptimum pHxe2x80x9d refers to a pH range in which relative active value is not less than 80% of the maximum active value, and the case in which pH 7.0 is included in such a pH range is referred to as xe2x80x9chaving optimum pH in a neutral pH rangexe2x80x9d in the present invention.
Similarly, the phrase xe2x80x9chaving optimum pH in a weak acidic rangexe2x80x9d in the present invention refers to a case in which pH 5.5 is included in the optimum pH range and the maximum active value exists in the acidic side.
For converting cholesterol in a cholesterol-containing substance to coprostanol via 4-cholesten-3-one and coprostan-3-one by utilizing the enzymatic actions of cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase sequentially in the present invention, any of an enzymatic conversion method using these enzymes and a microbial conversion method using microbial cells having these enzymatic activities can be used.
As the enzymatic conversion method, there is listed, for example, a method in which enzymes having activities of the above-mentioned cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase are added to a cholesterol-containing substance for converting cholesterol to coprostanol, and the enzyme which can be used in this enzymatic conversion method is not limited to a purified enzyme and may also be a crude enzyme which has not been purified but has enzymatic activity.
It is particularly desirable in this enzymatic conversion method to be sequentially treated with cholesterol dehydrogenase, the treatment with 4-cholesten-3-one dehydrogenase and the treatment with coprostan-3-one dehydrogenase continuously by successively-adding the enzymes and the like, from practical points of view such as simplification of the enzyme treatment process, improvement of conversion rate, and the like.
As the above-mentioned microbial conversion method, there is listed a method in which microbial cells having at least one enzymatic activity of cholesterol dehydrogenase activity, 4-cholesten-3-one dehydrogenase activity and coprostan-3-one dehydrogenase activity, or a treated material thereof are added to a cholesterol-containing substance for converting cholesterol to coprostanol, and it is desirable to use microbial cells having these enzymatic activities simultaneously or a treated material thereof. Further, a recombinant microbial cells having these enzymatic activities or a treated material thereof can also be used.
The material having the above-mentioned enzymatic activity (hereinafter, referred to as xe2x80x9cenzyme sourcexe2x80x9d) is usually added in the form of a powder or aqueous solution to a cholesterol-containing substance. Further, if necessary, coenzymes such as AND (P), AND (P) H and the like, enzymes such as phospholipase, lipase, protease and the like, or nicotinamide, phosphate ion and the like can be used alone or in combination together with the enzyme source.
In treatment for meat, for example, beef, pork, mutton or chicken, the enzyme source is mixed with minced meat, dispersed in sliced meat or injected in block meat. Further, the enzyme source can also be injected in blood vessels before and after slaughtering.
In treatment for milk, the enzyme source is added to milk, or milk is passed through a carrier to which the enzyme source is fixed. Further, in the case of fermented milk food, the enzyme source can also be added at milk fermentation.
In the treatment for eggs, the enzyme source is injected in the whole egg, or the enzyme source is mixed with yolk obtained by cracking the egg.
Further, when meat, milk, egg or seafood is cooked, the enzyme source may be added.
Regarding feeds for animals, poultry and pisciculture, it is possible to use a raw material for feed which has been treated with the enzyme source, or to mix the enzyme source in the process for preparing a feed.
In the above-mentioned enzymatic treatment, treatment conditions (temperature, time, pH) and the amount of enzyme to be added under which enzymatic conversion to coprostanol is possible are selected, and in general, the treatment is conducted at reaction temperature of 2 to 70xc2x0 C. and pH of 4 to 9 for 0.5 to 1xc3x97103 hours, and it is desirable to used a cholesterol reductase having optimum pH in the pH range possessed by the cholesterol-containing food itself to be treated in a practical point of view.
The amount used of each enzyme which is cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase or coprostan-3-one dehydrogenase to be used is from 1 to 1xc3x97105 units, preferably from 1xc3x97102 to 1xc3x97104 units per gram of cholesterol in food.
Optionally, AND(P) or AND(P)H which is a coenzyme of the above-mentioned enzyme can be added in an amount of 1xc3x9710xe2x88x924 to 2xc3x97102 g per gram of cholesterol.
Optionally, nicotinamide can also be added in an amount from 0.01 to 5% in terms of concentration in food. In meat which exhibits strong activity of decomposing these coenzymes it is preferable to added nicotinamide in an amount of 0.1% or more.
If required, a phosphate ion can also be added in an amount from 5 to 25 mM.
If occasion requires, it is also possible to add a phospholipase in an amount from 1 to 1xc3x97105 units per gram of a phospholipid and to add a lipase in an amount from 1 to 1xc3x97105 units per gram of a lipid.
Specific examples of the novel enzymes of the present invention include cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B. The method for production and the physical and chemical properties of these enzymes are described below.
The microorganism used in producing the novel cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A may be any microorganism having ability to produce the above-mentioned cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, and may also be a variant species or variant strain thereof. As a specific example of the microorganism having ability to produce the cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, for example, Eubacterium sp. CP 2 is listed.
The above-mentioned Eubacterium sp. CP 2 is a strain which has been newly separated from the feces of lions by the present inventors, and mycological properties thereof are as follows.
The present strain exhibits excellent growth by standing culture in a solution under anaerobic conditions at 37xc2x0 C. using the following basal medium. Tests regarding various properties were investigated under this condition.
Basal medium; 1% Casitone, 1% yeast extract, 0.5% soluble starch, 0.5% sodium pyruvate, 0.05% sodium thioglycolate, 0.05% calcium chloride, 0.0001% Resazurin, and 0.01% lecithin (pH 7.5)
(a) Morphological properties
{circle around (1)} Form of cell; short rod bacterium, and sometimes shown as almond figure.
{circle around (2)} Size of cell; 0.5 to 0.7 xcexcmxc3x970.7 to 1.0 xcexcm
{circle around (3)} Polymorphism; none
{circle around (4)} Mobility; none
{circle around (5)} spore; none
(b) Cultural properties
No growth was observed in bouillon plate agar-media and bouillon broth medium under aerobic or anaerobic conditions.
Cultural properties under a culturing condition and basal medium in which the present strain can grow are shown below.
{circle around (1)} Basal medium agar plate culture (culturing for 14 days)
i) Appearance of growth; forming weak small colony
ii) Color; transparent white
iii) Gloss; recognized
iv) Dispersible pigment; none
{circle around (2)} Basal medium solution culture (culturing for 3 days)
i) Growth on surface; none
ii) Turbidity; grown in the form of white emulsion at the bottom part
{circle around (3)} Gelatin stab culture in Basal medium
i) Condition of growth; excellent
ii) Liquefaction of gelatin; none
(c) Physiological properties in basal medium culture
{circle around (1)} Gram staining; positive
{circle around (2)} Reduction of nitrate; negative
{circle around (3)} Denitrification reaction; negative
{circle around (4)} MR test; negative
{circle around (5)} DVP test; negative
{circle around (6)} Generation of indole; negative
{circle around (7)} Generation of hydrogen sulfide; positive
{circle around (8)} Hydrolysis of starch; negative
{circle around (9)} Decomposition of esculin; positive
{circle around (10)} Utilization of an inorganic nitrogen source in a medium which has been obtained by removing 1% casitone and 1% yeast extract from the basal medium
i) Nitrate; negative
ii) Ammonium salt; negative
{circle around (11)} Generation of pigment; none
{circle around (12)} Urease; negative
{circle around (13)} Oxidase; negative
{circle around (14)} Catalase; negative
{circle around (15)} Growth range
i) Growth pH range; pH 6.0to pH 7.7 (optimum growth; around pH 7.3)
ii) Growth temperature range; 28 to 44xc2x0 C. (optimum growth temperature; around 35xc2x0 C.)
{circle around (16)} Attitude against oxygen; strictly anaerobic
{circle around (17)} O-F test (Hugh Leifson method); negative
{circle around (18)} Generation of acid and gas
i) L-arabinose; acid (none), gas (none)
ii) D-xylose; acid (none), gas (none)
iii) D-glucose; acid (observed), gas (none)
iv) D-mannose; acid (observed), gas (none)
v) D-fructose; acid (none), gas (none)
vi) D-galactose; acid (observed), gas (none)
vii) Maltose; acid (none), gas (none)
viii) Sucrose; acid (none), gas (none)
ix) Lactose; acid (none), gas (none)
x) Trehalose; acid (none), gas (none)
xi) D-sorbitol; acid (none), gas (none)
xii) D-mannitol; acid (none), gas (none)
xiii) Inositol; acid (none), gas (none)
xiv) Glycerin; acid (none), gas (none)
xv) Starch; acid (none), gas (none)
xvi) Metabolite from saccharides; butyric acid or acetic acid
(d) Other various properties; test according to An-IDENT
{circle around (1)} xcex1-glucosidase; positive
{circle around (2)} xcex2-glucosidase; positive
{circle around (3)} Alkali phosphatase; positive
{circle around (4)} xcex1-galactosidase; positive
{circle around (5)} Phenylalanineamino peptidase; negative
{circle around (6)} Decomposition of arginine; positive
{circle around (7)} Decomposition indoxyl acetate; negative
The present strain was of a gram positive strictly anaerobic short rod bacterium, did not form a spore, had no mobility, was negative against all of a catalase, oxidase and urease and formed acids from glucose and lactose, and main metabolite thereof was butyric acid or acetic acid. The classificational position of the strain having these mycological properties was compared with the description of Bergey""s Manual of Systematic Bacteriology vol. 2, 1986, as a result, the strain was identified as a bacterium belonging to Eubacterium, and the CP 2 strain was named as Eubacterium sp. CP 2. This strain was deposited in the name of FERM BP-5501 to National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan) on Apr. 12, 1996.
As the medium used for culturing a microorganism which produces cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A of the present invention, any of a synthetic medium or natural medium containing a carbon source, nitrogen source, inorganic substance and the like can be used.
As the carbon source, for example, various carbohydrates can be used such as a soluble starch, lactose, pyruvic acid, glucose, molasses and the like, and the amount used thereof is preferably from 1 to 20 g/L.
As the nitrogen source, for example, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium acetate, or nitrogen-containing organic compounds such as peptone, yeast extract, corn steep liquor, casein decomposed material, meat extract, and the like, can be used, and the amount used thereof is preferably from 1 to 20 g/L.
As the inorganic substance, for example, sodium chloride, calcium chloride, magnesium sulfate and the like are used, and the amount used thereof is preferably from 0.1 to 2 g/L. The amount used of a surfactant such as lecithin and the like is preferably from 0.01 to 1 g/L. The amount used of sodium thioglycolate is preferably from 0.1 to 1 g/L.
Culturing is conducted under an anaerobic condition by standing culture or stirring culture. Culturing temperature may advantageously be a temperature at which a microorganism grows and produces cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A, and preferably from 35 to 40xc2x0 C. Culturing period varies depending on conditions, and culturing may advantageously be conducted until the maximum amount of cholesterol dehydrogenase A, 4-cholesten-3-one dehydrogenase A and coprostan-3-one dehydrogenase A are produced, and usually from about 5 to 10 days.
Cholesterol dehydrogenase A that is produced by the CP 2 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction.
Cholesterol+NADPxe2x86x924-cholesten-3-one+NADPH
(b) Substrate specificity
The present enzyme reacts with steroid having a hydroxyl group at 3xcex2 position, and has relative activity of 38, 74, and 30 regarding xcex2 sitosterol, campesterol, stigmasterol when the activity of cholesterol is 100.
(c) Optimum pH: 6.5 to 7.8
(d) Stable pH: 5.3 to 9.0
An enzyme solution was allowed to stand still at 37xc2x0 C. for 15 minutes using various pH buffer solutions, and remaining activity was measured, and pH range in which activity was not less than 50% of the maximum active value was regarded as a stable pH range. (The following is the same.)
(e) Measurement of titer:
With 0.2 ml of 3 mM cholesterol micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM piperazine-N,Nxe2x80x2-bis-(2-ethane sulfuric acid) (hereinafter, referred to as xe2x80x9cPIPESxe2x80x9d) buffer solution (pH 7.5) containing 0.5% Triton X-100 and 1 mM dithiothreitol (hereinafter, referred to as xe2x80x9cDTTxe2x80x9d), and 0.1 ml of a 10 mM AND solution, the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of 4-cholesten-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of 4-cholesten-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of 4-cholesten-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing activity.
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM p-chloro mercury phenylsulfonate (PCMB), iodine acetamide and ethylenediamine tetraacetate (EDTA) are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 82 and 105 respectively, if the enzyme activity without adding inhibitor is defined as 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 95 and 3.2, respectively, in comparison with the case of no addition.
(i) Purification method:
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM MDTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10% glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the same buffer solution, then, adsorbed on Red Sepharose CL-6B (Pharmacia). Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADP) is used as a coenzyme.
(k) Molecular weight:
The molecular weight according to SDS polyacrylamide electrophoresis method is about 57,500.
The 4-cholesten-3-one dehydrogenase A of the present invention produced by the CP 2 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction.
4-cholesten-3-one+NADHxe2x86x92coprostan-3-one+AND
(b) Substrate specificity
The relative activities are 0, 41 and 0 for testosterone, progesterone and prognerone when the activity of 4-cholesten-3-one is defined as 100.
(c) Optimum pH: 5.4 to 6.5
(d) Stable pH: 5.5 to 7.2
(e) Measurement of titer:
With 0.2 ml of 3 mM 4-cholesten-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM PIPES buffer solution (pH 7.5) containing 0.5% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADH solution, the resulted mixture is added 0.05 ml of an enzyme solution, and reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of coprostan-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostan-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostan-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 6.0 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing the activity
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 47, 94 and 82 respectively, if the enzyme activity without adding inhibitor is defined as 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 110 and 0, respectively, in comparison to the case of no addition.
(i) Purification method:
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM MDTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10% glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the same buffer solution, then, adsorbed on Resource Column (Pharmacia). Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 1.0 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide (NADH) is used as a coenzyme.
(k) Molecular weight:
The molecular weight according to SDS polyacrylamide electrophoresis method is about 37,500.
The coprostan-3-one dehydrogenase A of the present invention produced by the CP 2 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction. coprostan-3-one+NADPHxe2x86x92coprostanol+NADP
(b) Substrate specificity
The relative activities are 13.2 for 5xcex1-cholesten-3-one when the activity of coprostan-3-one is defined as 100.
(c) Optimum pH: 5.2 to 5.7
(d) Stable pH: 4.0 to 7.5
(e) Measurement of titer:
With 0.2 ml of 3 mM coprostan-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 40 mM Britton-Robinson buffer solution (pH 6.0) containing 0.5% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADPH solution, the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction. The enzyme solution was mixed with equal amounts of activating fractions and left for 15 minutes at 5xc2x0 C., then used.
Then, the quantity of coprostanol produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostanol in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostanol per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing the activity.
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes in the presence of 0.5 mM NADPH, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 41, 82 and 101 respectively, if the enzyme activity without adding inhibitor is defined as 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 12 and 2.7, respectively, in comparison with the case of no addition.
(i) Purification method:
Method 1;
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0, containing 0.1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM PIPES buffer solution (pH 7.5, containing 1 mM DTT, 0.2% Triton X-100, 10% glycerol), then, passed through Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution, and fractions which have not been adsorbed on Blue Sepharose CL-6B are recovered, and dialyzed against a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0) (containing 0.5 mM NADPH, 1 mM DTT, 0.2% Triton X-100, 10% glycerol). The solution is adsorbed on DEAE-Cellulofine which has been equilibrated with the same buffer solution, then, the buffer solution flows with sodium chloride concentration being increased continuously from 0 to 1.0 M, and active fractions are collected and dialyzed against the buffer solution. The active fractions are adsorbed on Blue Sepharose CL-6B, and fractions which had not been adsorbed were recovered. The fractions which had not been adsorbed were heated at 95xc2x0 C. for 10 minutes to remove protein and to obtain activating fractions. Then, the active fractions which had been adsorbed on Blue Sepharose were eluted with the same buffer solution containing 1 mM NADPH with sodium chloride concentration being increased continuously from 0 to 1.0 M. To these active fractions, the above-mentioned activating fractions were added to prepare a purified sample.
Method 2;
Microbial cells are collected from the culture by centrifugal separation, and the cells are suspended in a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0, containing 0.1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 0.2% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. The crude enzyme solution is dialyzed against a 20 mM phosphoric acid-citric acid buffer solution (pH 6.0, containing 1 mM DTT, 0.2% Triton X-100, 30% glycerol), then, adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against a 20 mM phosphoric acid-citric acid buffer solution B (pH7.5, containing 1mM DTT, 0.2% Triton X-100, 30% glycerol), then adsorbed on DEAE-Cellulofine which has been equilibrated with the same buffer solution B, then, the buffer solution flows with sodium chloride concentration being increased continuously from 0 to 1.0 M, and active fractions are collected and dialyzed against the buffer solution. The active fractions are dialyzed against the same buffer solution B, then adsorbed on Resource Column (Pharmasia). Then, the above-mentioned buffer solution B flows with sodium chloride concentration being increased continuously from 0 to 1.0 M and active fractions are collected to prepare a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADPH) is used as a coenzyme.
(k) Molecular weight:
The molecular weight according to SDS polyacrylamide electrophoresis method is about 29,000.
Then, the method for producing the novel cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B is described below.
The microorganism used in producing the present enzymes may be any microorganism having the ability to produce the above-mentioned cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B, and may also be a variant species or variant strain thereof. As a specific example of the microorganism having the ability to produce the cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B and belonging to Eubacterium, for example, Eubacterium sp. CP 1 is listed.
The above-mentioned Eubacterium sp. CP 1 is a strain which has been newly separated from the feces of humans by the present inventors, and mycological properties thereof are as follows.
The present strain exhibits excellent growth by standing culture in a solution under anaerobic conditions at 37xc2x0 C. using the following basal medium. Tests regarding various properties were investigated under this condition.
Basal medium; 1.2% a bovine brain extracted lipid, 1.8% Trypticase, 0.05% yeast extract, 0.13% dipotassium phosphate, 0.22% sodium chloride, 0.05% cholesterol, 0.04% L-cystine, 0.03% sodium thioglycolate, 0.05% agar, and a small amount of 0.05% Methylene Blue (pH 7.2)
(a) Morphological properties
{circle around (1)} Form of cell; long rod bacterium and grown in the form of chain
{circle around (2)} Size of cell; 0.3 to 0.5 xcexcmxc3x973.0 to 5.0 xcexcm
{circle around (3)} Polymorphism; none
{circle around (4)} Mobility; none
{circle around (5)} Spore; none
(b) Cultural properties
No growth was observed in the bouillon plate agar-media and bouillon broth media under aerobic or anaerobic conditions.
Cultural properties under culturing condition and basal medium in which the present strain can grow are shown below.
{circle around (1)} Basal medium agar plate culture (culturing for 21 days)
i) Appearance of growth; forming powdery colony
ii) Color; white
iii) Gloss; none
iv) Dispersible pigment; none
{circle around (2)} Basal medium solution culture (culturing for 5 days)
i) Growth on surface; none
ii) Turbidity; grows, but, discrimination from sediment in a medium by turbidity is difficult
{circle around (3)} Gelatin stab culture in basal medium
i) Condition of growth; excellent
ii) Liquefaction of gelatin; none
(C) Physiological properties in basal medium culture
{circle around (1)} Gram staining; positive
{circle around (2)} Reduction of nitrate; negative
{circle around (3)} Litmus milk; coagulation negative
{circle around (4)} Generation of indole; negative
{circle around (5)} Generation of hydrogen sulfide; negative
{circle around (6)} Hydrolysis of starch; positive
{circle around (7)} Decomposition of esculin; positive
{circle around (8)} Utilization of an inorganic nitrogen source in a medium which has been obtained by removing trypticase and yeast extract from a basal medium
i) Nitrate; negative
ii) Ammonium salt; negative
{circle around (9)} Generation of pigment; none
{circle around (10)} Urease; negative
{circle around (11)} Oxidase; negative
{circle around (12)} Catalase; negative
{circle around (13)} Growth range
i) Growth pH range; pH 5 to pH 8 (optimum growth; around pH 7)
ii) Growth temperature range; 33 to 40xc2x0 C. (optimum growth temperature; around 34xc2x0 C.)
{circle around (14)} Behavior against oxygen; strictly anaerobic
{circle around (15)} O-F test (Hugh Leifson method); negative
{circle around (16)} Generation of acid
i) L-arabinose; acid (small amount)
ii) D-xylose; acid (observed)
iii) D-glucose; acid (observed)
iv) D-mannose; acid (observed)
v) D-fructose; acid (small amount)
vi) D-galactose; acid (observed)
vii) maltose; acid (observed)
viii) Sucrose; acid (small amount)
ix) Lactose; acid (observed)
x) Trehalose; acid (none)
xi) D-sorbitol; acid (none)
xii) D-mannitol; acid (none)
xiii) Inositol; acid (none)
xiv) Glycerin; acid (none)
xv) Starch; acid (none)
xvi) Ribose; acid (observed)
xvii) Cellobiose; acid (observed)
xviii) Lactose; acid (observed)
xix) Melibiose; acid (observed)
xx) Raffinose; acid (observed)
xxi) Salicin; acid (observed)
xxii) Amygdalin; acid (observed)
xxiii) Melezitose; acid (none)
xxiv) Glycogen; acid (none)
xxv) Inulin; acid (none)
xxvi) Metabolite from saccharides; acetic acid
The present strain was gram positive strictly anaerobic long rod bacterium, did not form spores, had no mobility, was negative against all of a catalase, oxydase and urease and formed acids from glucose, and main metabolite thereof was acetic acid. The classificational position of the strain having these mycological properties was compared with description of Bergey""s Manual of Systematic Bacteriology vol. 2, 1986, as a result, the strain was identified as a bacterium belonging to Eubacterium, and the CP 1 strain was named as Eubacterium sp. CP 1. This strain was deposited in the name of FERM BP-5500 to National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture, Japan) on Apr. 12, 1996.
As the medium used for culturing a microorganism which produces cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B of the present invention, any of the synthetic mediums or natural mediums containing a carbon source, nitrogen source, inorganic substance and the like can be used.
As the carbon source, for example, various carbohydrates can be used such as glucose, maltose, molasses and the like, and the amount used thereof is preferably from 1 to 20 g/L.
As a nitrogen source, for example, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium acetate, or nitrogen-containing organic compounds such as peptone, yeast extract, corn steep liquor, casein decomposed material, meat extract, and the like, can be used, and the amount used thereof is preferably from 1 to 20 g/L. The amount of solvent-extract used (chloroform: methanol=2:1) of a bovine brain lipid is preferably from 1 to 20 g/L.
As an inorganic substance, for example, sodium chloride, calcium chloride, magnesium sulfate, potassium monohydrogen phosphate and the like are used, and the amount used thereof is preferably from 0.1 to 2 g/L. The amount used of cystine and sodium thioglycolate is preferably from 0.1 to 1 g/L.
Culturing is conducted under anaerobic conditions by standing culture or stirring culture. Culturing temperature may advantageously be a temperature at which a microorganism grows and produces cholesterol dehydrogenase B, 4-cholesten-3-one dehydrogenase B and coprostan-3-one dehydrogenase B, and preferably from 35 to 40xc2x0 C. Culturing period varies depending on conditions, and culturing may advantageously be conducted until the maximum amount of these enzymes are produced, usually from about 3 to 10 days.
Cholesterol dehydrogenase B of the present invention that is produced by the CP 1 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction.
Cholesterol+NADPxe2x86x924-cholesten-3-one+NADPH
(b) Substrate specificity
The present enzyme reacts with steroid having a hydroxyl group at 3xcex2 position, and has relative activity of 67, 50 and 39 regarding xcex2 sitosterol, campesterol, stigmasterol when the activity of cholesterol is defined as 100.
(c) Optimum pH: 6.7 to 7.7
(d) Stable pH: 5.0 to 10.5
(e) Measurement of titer:
With 0.2 ml of 3 mM cholesterol micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 50 mM phosphoric acid buffer solution (pH 7.5) containing 1.0% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADP solution, to the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of 4-cholesten-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of 4-cholesten-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of 4-cholesten-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperatures:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing activity.
(g) Range of temperature stability
After heating treatment at 40xc2x0 C. for 10 minutes ,it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetic acid and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 98 and 102 respectively, if the enzyme activity without adding inhibitor is 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are both 0 in comparison to the case of no addition.
(i) Purification method:
Air is bubbled through the culture, and microbial cells adsorbed on the bubble are recovered and concentrated. The concentrated solution is subjected to centrifugal separation to collect the cells, and the cells are suspended in a 20 mM phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 1.0% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and the mixture is dialyzed against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM PMSF). The crude enzyme solution is adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution containing 0.25% Triton X-100. Then, lipid components are eluted with the same buffer solution containing 1.0% Triton X-100, then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the above-mentioned buffer solution, then, adsorbed on DEAE-Sepharose Fast Flow which has been equilibrated with the same buffer solution containing 1.0% Triton X-100. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 0.5 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADP) is used as a coenzyme.
The 4-cholesten-3-one dehydrogenase B of the present invention produced by the CP 1 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction. 4-cholesten-3-one+NADHxe2x86x92coprostan-3-one+AND
(b) Substrate specificity
The relative activities are 0 for 5-cholestenon-3-one when the activity of 4-cholesten-3-one is defined as 100.
(c) Optimum pH: 5.3 to 7.0
(d) Stable pH: 5.2 to 8.0
(e) Measurement of titer:
With 0.2 ml of 3 mM 4-cholesten-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 1.0% Triton X-100and 1 mM DTT, and 0.1 ml of a10 mM NADH solution, the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of coprostan-3-one produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostan-3-one produced in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostan-3-one per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperatures:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 40xc2x0 C. is attended by increasing the activity.
(g) Range of temperature stability
After heating treatment at 37xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 99 and 125, respectively, if the enzyme activity without adding inhibitor is 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 107 and 0, respectively, in comparison with the case of no addition.
(i) Purification method:
Air is bubbled through the culture, and microbial cells adsorbed on the bubble are recovered and concentrated. The concentrated solution is subjected to centrifugal separation to collect the cells, and the cells are suspended in a 20 mM phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 1.0% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM PMSF, and the mixture was dialyzed against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM PMSF). The enzyme solution is adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution containing 0.25% Triton X-100. Then, lipid components are eluted with the same buffer solution containing 1.0% Triton X-100, then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulted active fractions are dialyzed against the above-mentioned buffer solution, then, adsorbed on DEAE-Sepharose Fast Flow which has been equilibrated with the same buffer solution containing 1.0% Triton X-100. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 0.5M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide (NADH) is used as a coenzyme.
The coprostan-3-one dehydrogenase B of the present invention produced by the CP 1 strain is a novel enzyme, and the physical and chemical properties and method for purification thereof are as follows.
(a) Action: it catalyses the following reaction. coprostan-3-one+NADPHxe2x86x92coprostanol+NADP
(b) Substrate specificity
The relative activities are 0 for 5xcex1-cholesten-3-one when the activity of coprostan-3-one is defined as 100.
(c) Optimum pH: 5.4 to 7.6
(d) Stable pH: 4.5 to 7.0
(e) Measurement of titer:
With 0.2 ml of 3 mM coprostan-3-one micelle solution containing 0.33% Triton X-100 is mixed 0.3 ml of a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 1.0% Triton X-100 and 1 mM DTT, and 0.1 ml of a 10 mM NADPH solution, to the resulted mixture is added 0.05 ml of an enzyme solution, reacted for 30 minutes at 37xc2x0 C., and then added 0.1 ml of chloroform to extract sterol and to terminate the reaction.
Then, the quantity of coprostanol produced in the reaction solution is determined using TLC/FID iatroscan. The quantity of coprostanol in the reaction solution is determined likewise using an inactivated enzyme which has been previously heated as a control. The enzyme activity which produces 1 xcexcmol of coprostanol per one minute is regarded as 1 unit.
(f) Range of suitable reaction temperature:
In reaction at pH 7.5 for 30 minutes, increasing temperature up to 45xc2x0 C. is attended by increasing the activity.
(g) Range of temperature stability
After heating treatment at 45xc2x0 C. for 10 minutes, it keeps activity of not less than 80% of that before the treatment.
(h) Influence of inhibitor, metal ion:
When 1 mM PCMB, iodine acetamide and EDTA are added and reacted for 30 minutes at pH 7.5 and 37xc2x0 C., the relative activities are 0, 95 and 112, respectively, if the enzyme activity without adding inhibitor is 100. Further, when the present enzyme is reacted in the presence of 1 mM iron chloride and copper chloride, the relative activities are 105 and 0, respectively, in comparison to the case of no addition.
(i) Purification method:
Air is bubbled through the culture, and microbial cells adsorbed on the bubbles are recovered and concentrated. The concentrated solution is subjected to centrifugal separation to collect the cells, and the cells are suspended in a 20 mM phosphoric acid buffer solution (pH 7.5, containing 1 mM DTT). The cells are disrupted by ultrasonication, further, treated with ultrasonication in the presence of 1.0% Triton X-100, solid components are removed by centrifugal separation, to obtain a crude enzyme solution. To the crude enzyme solution is added 1 mM EDTA, 1 mM iodine acetic acid and 0.5 mM PMSF, and the mixture is dialyzed against a 20 mM Tris-hydrochloric acid buffer solution (pH 7.5, containing 1 mM DTT, 10% glycerol, 1 mM EDTA, 1 mM iodine acetic acid, and 0.5 mM PMSF). The crude enzyme solution is adsorbed on Blue Sepharose CL-6B (Pharmacia) which has been equilibrated with the same buffer solution containing 0.25% Triton X-100. Then, lipid components are eluted with the same buffer solution containing 1.0% Triton X-100, then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 3.0 M, and active fractions are collected. The resulting active fractions are dialyzed against the above-mentioned buffer solution, then, adsorbed on DEAE-Sepharose Fast Flow which has been equilibrated with the same buffer solution containing 1.0% Triton X-100. Then, the above-mentioned buffer solution flows with sodium chloride concentration being increased continuously from 0 to 0.5 M, and active fractions are collected to obtain a purified sample.
(j) Coenzyme:
Reduced type xcex2-nicotinamide adenine dinucleotide phosphoric acid (NADPH) is used as a coenzyme.
As the cholesterol-reduced composition of the present invention, microbial cells or treated materials thereof, crude purified enzymes, purified enzymes and the like containing these three enzymes may be used without any treatment provided they have activities of a cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase, and further, those in the form of tablets, powders, fine particles, granules, capsules, syrups and the like molded with vehicles which are acceptable for food or medicine may be used. The composition of the present invention may be added as a composition to be added into food or feed for reducing the amount of cholesterol in the food or feed, or may be orally administered via oral route for reducing the cholesterol level in serum. When a crude purified enzyme or purified enzyme is used as the composition of the present invention, the composition optionally may be advantageously prepared so that nicotinamide, phosphate ion or phospholipase is contained in the composition. Examples of the form of the oral composition of the present invention include tablets, powders, fine particles, granules, capsules, syrups, enteric agent, troches and the like. In the case of addition or administration, as the vehicle, any compound such as saccharides like sorbitol, lactose, glucose, lactose, dextrin, starch, crystalline cellulose and the like; inorganic compounds like calcium carbonate, calcium sulfate and the like; distilled water, sesame oil, corn oil, olive oil, cotton seed oil and the like, generally can be used. In preparing the composition, additives such as binder, lubricant, disperser, suspending agent, emulsifying agent, diluent, buffering agent, antioxidant, bacterium inhibiting agent and the like may be used.
The amount to be added may be advantageously controlled to be the above-mentioned value necessary for the enzymatic conversion or the microbial conversion in the method for producing a cholesterol-reduced substance of the present invention.
The dosage varies depending on age, sex, administration pattern, times of administration, form and the like, and regarding dosage for oral administration for adults, it is suitable that the amount of bacteria is 1xc3x97107 to 1xc3x971012 cell/day, preferably 1xc3x97108 to 1xc3x971011 cell/day and the amount of three enzymes is 10 to 1xc3x97105 unit/day, preferably 1xc3x97102 to 1xc3x97104 unit/day, and that the composition is divided into 1 to 4 portions for one day-administration. If necessary, dosage out of the above-mentioned restriction can also be adopted.